ABSTRACT: The goal of this project is to understand the mechanism by which the Rho/MLCP (myosin light chain phosphatase) pathway regulates myosin light chain (MLC) phosphorylation and contraction in vascular smooth muscle. In the present proposal, we will clarify the function of p116Rip, the novel regulatory protein of the RhoA/MLCP pathway, based upon our recent findings of this MBS/RhoA binding molecule. While a number of studies have been done on the regulatory role of Rho kinase in smooth muscle, nothing is known about the function of PKN, another RhoA down-stream kinase. Our PRELIMINARY STUDIES raise the possibility that PKN plays a role in the regulation of the RhoA/MLCP pathway, and we will study the function and regulation of PKN in the agonist induced regulation of smooth muscle contraction. Based upon our PRELIMINARY STUDIES, we propose the following hypothesis of the regulatory function of PKN and p116Rip. Upon agonist stimulation, RhoA translocates to the membrane and cytosolic PKN, which has a binding affinity for active RhoA, is recruited to the membrane. PKN becomes activated and sustains the membrane binding of RhoA, thus prolongs RhoA activity. On the other hand, p116Rip associates with myosin and MBS at the actomyosin fiber and activates MLCP reaction, thus facilitating MLC dephosphorylation. P116Rip, at the actomyosin filaments, interacts with cytosolic RhoA to prevent translocation to the membrane, thus attenuating RhoA activation. These effects result in the increase in MLCP activity and down-regulation of MLC phosphorylation in smooth muscle. In this proposal, we will verify this hypothesis. We will first use a siRNA approach to eliminate p116Rip and PKN, respectively, and study the effects of the specific siRNAs in the agonist induced change in MLC20 phosphorylation and muscle contraction. Once we identify the function of p116Rip and PKN in the regulation of MLC20 phosphorylation, we will study the regulatory mechanism of p116Rip and PKN activity. It has been known that the activation of Rho and its downstream molecules are closely related to the translocation of these molecules to the membrane. To further address our hypothesis, we will study the spatio-temporal change in the localization of these molecules after stimulation by using 3D digital imaging analysis of the differentiated smooth muscle cells. To achieve this, we will introduce fluorescent protein (FP)-tagged regulatory proteins using the protein delivery technique. We will also use our newly developed total internal reflection fluorescence (TIRF) microscope to monitor the change in the near-membrane domain of the cells. Furthermore, we will study the binding of p116Rip and PKN with their target molecules by using FRET analysis, thus monitoring the spatio-temporal change in the interaction of the molecules in living cells. The proposed project will clarify the mechanism by which agonists induce vascular smooth muscle contraction, which should provide novel insights into the regulation of vascular constriction and contribute to the pathogenesis of cardiovascular diseases.